Development of curative therapies for Ewing sarcomas by interdisciplinary cooperative groups in Europe.

Curative therapies for Ewing sarcoma have been developed within cooperative groups. Consecutive clinical trials have systematically assessed the impact and timing of local therapy and the activity of cytotoxic drugs and their combinations. They have led to an increase of long-term disease-free survival to around 70% in patients with localized disease. Translational research in ES remains an area in which interdisciplinary and international cooperation is essential for future progress. This article reviews current state-of-the art therapy, with a focus on trials performed in Europe, and summarizes novel strategies to further advance both the cure rates and quality of survival.

Pelvic Ewing sarcomas. Three-dimensional conformal vs. intensity-modulated radiotherapy.

PURPOSE: The goal of the present work was to assess the potential advantage of intensity-modulated radiotherapy (IMRT) over three-dimensional conformal radiotherapy (3D-CRT) planning in pelvic Ewing's sarcoma. PATIENTS AND METHODS: A total of 8 patients with Ewing sarcoma of the pelvis undergoing radiotherapy were analyzed. Plans for 3D-CRT and IMRT were calculated for each patient. Dose coverage of the planning target volume (PTV), conformity and homogeneity indices, as well as further parameters were evaluated. RESULTS: The average dose coverage values for PTV were comparable in 3D-CRT and IMRT plans. Both techniques had a PTV coverage of V95 > 98 % in all patients. Whereas the IMRT plans achieved a higher conformity index compared to the 3D-CRT plans (conformity index 0.79 ± 0.12 vs. 0.54 ± 0.19, p = 0.012), the dose distribution across the target volumes was less homogeneous with IMRT planning than with 3D-CRT planning. This difference was statistically significant (homogeneity index 0.11 ± 0.03 vs. 0.07 ± 0.0, p = 0.035). For the bowel, Dmean and D1%, as well as V2 to V60 were reduced in IMRT plans. For the bladder and the rectum, there was no significant difference in Dmean. However, the percentages of volumes receiving at least doses of 30, 40, 45, and 50 Gy (V30 to V50) were lower for the rectum in IMRT plans. The volume of normal tissue receiving at least 2 Gy (V2) was significantly higher in IMRT plans compared with 3D-CRT, whereas at high dose levels (V30) it was significantly lower. CONCLUSION: Compared to 3D-CRT, IMRT showed significantly better results regarding dose conformity (p = 0.012) and bowel sparing at dose levels above 30 Gy (p = 0.012). Thus, dose escalation in the radiotherapy of pelvic Ewing's sarcoma can be more easily achieved using IMRT.

Homophilic adhesion between Ig superfamily carcinoembryonic antigen molecules involves double reciprocal bonds.

Both carcinoembryonic antigen (CEA) and neural cell adhesion molecule (NCAM) belong to the immunoglobulin supergene family and have been demonstrated to function as homotypic Ca(++)-independent intercellular adhesion molecules. CEA and NCAM cannot associate heterotypically indicating that they have different binding specificities. To define the domains of CEA involved in homotypic interaction, hybrid cDNAs consisting of various domains from CEA and NCAM were constructed and were transfected into a CHO-derived cell line; stable transfectant clones showing cell surface expression of CEA/NCAM chimeric-proteins were assessed for their adhesive properties by homotypic and heterotypic aggregation assays. The results indicate that all five of the Ig(C)-like domains of NCAM are required for intercellular adhesion while the COOH-terminal domain containing the fibronectin-like repeats is dispensable. The results also show that adhesion mediated by CEA involves binding between the Ig(V)-like amino-terminal domain and one of the Ig(C)-like internal repeat domains: thus while transfectants expressing constructs containing either the N domain or the internal domains alone were incapable of homotypic adhesion, they formed heterotypic aggregates when mixed. Furthermore, peptides consisting of both the N domain and the third internal repeat domain of CEA blocked CEA-mediated cell aggregation, thus providing direct evidence for the involvement of the two domains in adhesion. We therefore propose a novel model for interactions between immunoglobulin supergene family members in which especially strong binding is effected by double reciprocal interactions between the V-like domains and C-like domains of antiparallel CEA molecules on apposing cell surfaces.

Preparation of synthetic polypeptide domains of carcinoembryonic antigen and their use in epitope mapping.

Genes encoding the four principal polypeptide domains (N, A1-B1, A2-B2, and A3-B3) of carcinoembryonic antigen (CEA) were synthesized and expressed in Escherichia coli as fusion products with bacterial CMP-KDO synthetase (CKS). The four synthetic fusion proteins were purified in high yield and used as targets in Western blots for 11 anti-CEA MAbs and to compete with immobilized CEA for binding to four of these MAbs. Each of the MAbs showed strong binding to one or more of the fusion proteins. In Western blots, MAbs H19C91 and 4230 bound only to CKS-N. MAbs H8C2 and H11C35 bound only CKS-A1-B1, and MAbs T84.66, H46C136, and H21C83 appeared to be specific for CKS-A3-B3. None of the MAbs tested bound only to CKS-A2-B2. However, two MAbs bound both CKS-A1-B1 and CKS-A3-B3 and one MAb (3519) bound to all three of the repeated domains. Since these three domains exhibit over 90% amino acid sequence homology, the latter results were not surprising. The competition studies largely confirmed the results of Western blots but did show some MAb-fusion protein interactions not observed in Western blots. These competition studies also allowed estimation of the relative affinities of the MAbs for the synthetic domains and for native CEA. These studies demonstrated that epitopes in CEA recognized by the MAbs in this study are peptide in nature and that the fusion proteins are of utility in the localization of the epitopes on the polypeptide chain of CEA.

FokI method of gene synthesis.

An accurate, fast and simple method is presented for synthesis of a gene, or any DNA fragment with a defined sequence. The method is based on the observation that large (approx. 100 bp long) inserts can be cloned into a plasmid using a technique of oligodeoxynucleotide (oligo)-directed double-strand (ds) break repair. The procedure involves transformation of Escherichia coli with a denatured mixture of an insert-carrying oligo and linearized plasmid DNA [Mandecki, Proc. Natl. Acad. Sci. USA 83 (1986) 7177-7181]. The nucleotide (nt) sequences are inserted between two FokI restriction nuclease sites in one of four pUC-derived plasmids. Since FokI makes a staggered ds break at a DNA site 9 and 13 nt away from its recognition site, upon cleavage of the plasmid DNA with FokI, a restriction fragment is liberated that by design contains unique 4-nt-long 5'-protruding ends. The uniqueness of ends permits efficient and directed simultaneous ligation of several restriction fragments to form a gene. The method offers flexibility due to the modular-type assembly and does not require any restriction sites within the constructed gene. The sequence error rate is low: about one error per 4000 bp of DNA cloned. Synthetic DNA for only one DNA strand needs to be provided. The method was applied to the synthesis of a gene fragment encoding the N-terminal 143 amino acid residues of the human immunodeficiency virus transmembrane protein (p41).

An Escherichia coli expression vector for high-level production of heterologous proteins in fusion with CMP-KDO synthetase.

The construction of a vector which overproduces the enzyme, CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyl-transferase (CMP-KDO synthetase or CKS) and its use as an expression vector for producing heterologous proteins in E. coli is described. The vector, which includes a modified lac promoter and synthetic ribosome binding site upstream of the native kdsB gene (encoding CKS), produces CKS at levels as high as 70% of the total cellular proteins. Several heterologous gene sequences have been fused to the 3'-end of the kdsB gene with resulting protein fusions produced at a level of up to 40% of the total cellular proteins.

Mouse monoclonal antibody 5-21-3 recognizes a contiguous, conformation-dependent epitope and maps to a hydrophilic region in HIV-1 gp41.

Mouse monoclonal antibody 5-21-3 is mapped to an epitope within a hydrophilic region of HIV-1 gp41 between amino acids 642 and 665 (numbering by Meyers et al. based on HXB2 isolate). The epitope is formed from amino acids within the sequence IHSLIEESQNQQEKNEQELLELDK; however, antibody 5-21-3 is unable to recognize the epitope-forming sequence when it is presented to the antibody in the form of a short (642-665) synthetic polypeptide. The epitope apparently is partially formed when additional native sequence of varying length is added to the amino and/or carboxy ends of the epitope-forming sequence, and 5-21-3 binds these larger synthetic polypeptides to varying degrees depending on the position and length of the flanking sequences. The 5-21-3 epitope apparently is formed from contiguous amino acids which require a specific, conformation-dependent, secondary structure for proper epitope formation. Binding preferences exhibited by 5-21-3 toward synthetic polypeptides and recombinant proteins may reflect the conformational nature of the epitope in disrupted HIV which elicited formation of the monoclonal.

Time- and dose-dependent changes of intracellular cytokine and cytokine receptor profile of Ewing tumour subpopulations under the influence of ionizing radiation.

PURPOSE: Cytokines and their corresponding cell surface receptors are involved in intercellular signalling pathways and in the radioresistance of normal and malignant cells. The aim was the characterization of the expression of intracellular cytokines, their receptors and apoptosis-associated markers under the influence of radiation. MATERIAL AND METHODS: Two Ewing tumours were characterized in vitro before and 4, 24 and 72 h after radiation with 5 and 10 Gy, and in vivo 4, 6 and 15 days after radiation with 5 and 30 Gy by five parameter flow cytometry. Direct fluorescence-conjugated antibodies directed against intracellular cytokines (interferon-gamma, tumour necrosis factor [TNF]-alpha, interleukin 1) and their receptors (CD119, CD120a, CD121a) were used. Annexin V and 7-amino-actinomycin D were used to identify radiation-induced apoptosis. RESULTS: Inter- and intra-individual heterogeneities were identified by the expression of cytokine receptors and the intracellular cytokine profile before radiation. Time- and dose-dependent up-regulation of the cytokines TNF-alpha and interleukin 1 were found in vitro. In vivo, an up-regulation of CD120a and CD121a was detectable on tumour cell subpopulations. For interferon-gamma and CD119, no changes were seen. CONCLUSIONS: The observed radiation-induced changes of cytokine and receptor profile are an indication for complex intercellular interactions in view of radioresistance-associated mechanisms between cell populations within one individual tumour. The observed heterogeneous response on radiation might have therapeutic implications for an individualized therapy based on combined radiation and cytokine modulation, defined by flow cytometric characterization of markers potentially informative for radioresistance.

Effect of radiation on Ewing tumour subpopulations characterized on a single-cell level: intracellular cytokine, immunophenotypic, DNA and apoptotic profile.

PURPOSE: Adhesion molecules, cytokines and their corresponding cell-surface receptors are involved in intercellular signalling pathways, radioresistance and metastasis-mediating mechanisms of malignant cells. The aim was the characterization of changes in the marker profile of Ewing tumour cell subpopulations under the influence of radiation. MATERIALS AND METHODS: Three Ewing tumours were characterized in vitro and in vivo in a xenograft model before and after radiation by five-parameter flow cytometry. Antibodies directed against cell surface and intracellular antigens, apoptosis-associated markers and the DNA dye 7-aminoactinomycin D were used. RESULTS: Tumour cell subpopulations were identified by expression of adhesion molecules and cytokine receptors, intracellular cytokines, apoptotic markers and DNA content. Heterogeneous changes of flow cytometric profile were identified on tumour cell subpopulations after radiation. CONCLUSIONS: The changed profile of tumour cells under radiation might be associated with biological changes of tumour subpopulations in view of radioresistance and metastatic potential and might be useful to identify intercellular regulation mechanisms and to define parameters being predictive for a response to therapy.

Management of bone tumours in paediatric oncology.

The management of bone tumours in paediatric oncology requires careful multidisciplinary planning due to the need for multimodal therapy approaches. The non-specific symptoms often lead to a delayed definitive diagnosis of a bone tumour. Imaging procedures are of major importance for an individualised and optimised treatment planning. They have to be carried out before any surgery, including biopsies. The introduction of multi-agent chemotherapy has led to a significant improvement in survival rates in patients suffering from Ewing's sarcomas and osteosarcomas. However, local therapy still remains indispensable in order to achieve long-term survival. For osteosarcoma, surgery remains the only adequate local therapy modality. Radiotherapy may be considered if surgery is not feasible. In these cases, high radiation doses need to be applied. The choice for local therapy modality is not as clear in patients with Ewing's sarcoma. Today, surgery is often preferred if a wide or at least marginal resection can be carried out. Additional radiotherapy is advised in patients with marginal/intralesional resection or poor histological response to induction chemotherapy. Definitive radiotherapy is recommended for inoperable lesions. In the future, new radiotherapy approaches, such as intensity-modulated radiotherapy or proton therapy, may yield better results with minor risks of late effects.

Pediatric radiation oncology in Germany: a study of availability and application.

BACKGROUND: Radiotherapy plays a pivotal role in many multimodal therapy concepts in pediatric oncology. However, the absolute number of irradiated children is estimated to be quite low. The aim of this study was to evaluate the availability and application of pediatric radiation oncology in Germany. METHOD: In summer 2007, a standardized questionnaire was sent to all radiotherapy facilities in Germany. The questions regarded the structure of the departments, the number of irradiated children each year including the distribution of the different diagnoses, the number of curative treatments, inclusion in study trials, and existence of special contact persons for pediatric radiotherapy as well as technical aspects of irradiation of children. RESULTS: Answers to the questionnaires were obtained from 171 departments (77.4%). Of these, 67 (39%) stated to regularly treat children. These departments treated one to nine children in median each year (<5 children/year: 23 departments; >or=20 children: 15 departments). Most of these children suffered from brain tumors, Hodgkin's disease and acute lymphatic leukemia (ALL). Three-dimensional conformal radiotherapy was the most frequent treatment technique; special techniques like intensity-modulated radiotherapy (IMRT) or brachytherapy were rare. CONCLUSIONS: Due to quite low patient numbers treated in most radiotherapy facilities, individual experiences in pediatric radiation oncology can be assumed to be quite limited. As radiotherapy is part of multimodal therapy approaches in pediatric oncology and children treated with radiotherapy are at special risk for potential side effects, pediatric radiation oncology remains a sophisticated area. Therefore radiotherapy reference-institutions implemented by the therapy optimizing protocols are of fundamental importance.

Thyroid function in paediatric and young adult patients after sarcoma therapy: a report from the Late Effects Surveillance System.

OBJECTIVE: The role of chemotherapy in thyroid sequelae after cancer treatment has not been studied systematically, especially in sarcoma patients. The aim of this study was to determine the incidence of post-therapeutic thyroid disorders and their contributing factors in a cohort of paediatric sarcoma patients. DESIGN: Late effects of sarcoma treatment have been collected prospectively within the Late Effects Surveillance System (LESS) in Germany, Austria and Switzerland since 1998. PATIENTS: We studied 340 relapse-free paediatric patients (median age at diagnosis 12.2 [interquartile range (IQR) = 7.3-15.6 years] treated for osteosarcoma, soft tissue sarcoma or Ewing's sarcoma within the COSS-96, CWS-96/CWS-2002P or EICESS-92/EURO-E.W.I.N.G.-99 therapy trials. In addition to polychemotherapy, 127 patients were irradiated (mean cumulative dose 47 +/- 9.7 Gy), including 51 patients with irradiation to the head/neck region. Median follow-up was 24.6 (IQR = 11.9-44.9) months. MEASUREMENTS: We reviewed the results of yearly examinations of serum TSH and fT4 levels and thyroid ultrasound examinations. RESULTS: The incidence of thyroid disorders was 37% (19/51, 95% CI 24-52%) in patients with head/neck irradiation, and 11% (32/289, 95% CI 8-15%) in patients without irradiation to the head/neck. Thyroid disorders were more frequent in patients treated with idarubicin (P = 0.027) and trofosfamide (P = 0.016). We also found a significant association between raised TSH levels and treatment with trofosfamide (P = 0.008) or idarubicin (P = 0.037) (n = 250). CONCLUSIONS: The incidence of thyroid disorders in the head/neck-irradiated group was high. Even without head/neck irradiation, we found an increased proportion of patients with thyroid disorders, possibly as a result of chemotherapy.

[Register for the evaluation of side effects after radiation in childhood and adolescence--first results]. / Register zur Erfassung von Spätfolgen nach Strahlentherapie im Kindes- und Jugendalter--erste Ergebnisse.

BACKGROUND: Late effects after radiotherapy in childhood and adolescence have mainly been characterized retrospectively with small patient numbers. Therefore the German Group of Pediatric Radiation Oncology (APRO) established the "RegIster for the evaluation of late Side effects after radiation in childhood and adolescence" (RiSK). After a pilot phase starting in 2001 documentation has been performed all over Germany since 2004. This analysis shows the first results of "RiSK". PATIENTS AND METHODS: Radiation parameters including detailed organ doses as well as toxicity evaluations were collected prospectively from centers all over Germany in the study center. Standardized documentation forms were used. Documentation is planned for all children who receive radiotherapy in one of the German pediatric therapy trials. RESULTS: Until December 31st 2006, 696 documentations of radiotherapy and 526 acute as well as 836 late follow-up documentation forms have been collected. Altogether, 41 patients with late grade 3 and 16 patients with late grade 4-side effects were identified. Side effects mainly concerned joints with functional impairment (after combined radiotherapy and surgery), the bowel, skin and subcutis as well as blood parameters under continued chemotherapy. Patients with late side effects of a higher grade were mainly treated for Ewing's or soft tissue sarcomas (n=235 patients), representing 33.8% of all patients in this study. CONCLUSION: Fortunately, up to now only a few late grade 3 or 4 side effects of radiotherapy are shown for almost 700 documented patients. For further results, especially for the characterization of dose-effect-relationships, this study has to be continued with a higher patient number and a longer follow-up.

Heterogeneity of radiation induced apoptosis in Ewing Tumor cell lines characterized on a single cell level.

The objective of this study was to investigate heterogeneity of radiation induced apoptosis on a single cell level. Two Ewing tumor cell lines were characterized in vitro before and 24 and 72 h after radiation with 5 Gy by multiparametric flow cytometry. Annexin V, 7-AAD and fluorescence conjugated antibodies that were directed against HLA-ABC, CD11a and CD62L were used. Based on these markers radiation induced apoptosis was quantified, multiple apoptotic subpopulations were identified and a characteristic individual apoptotic profile was characterized. The characterization of HLA-ABC, CD11a and CD62L was informative to detect subpopulations of apoptotic cells. The observed heterogeneity and the identification of multiple apoptotic subpopulations reflect the complexity and diversity of biology of radiation induced cell death. This might be an indication for co-existing apoptotic pathways or it might represent sequential steps of the apoptotic cascade.

Primary structure of CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) from Escherichia coli.

The gene coding for CTP:CMP-3-deoxy-D-mannooctulosonate cytidylyltransferase (CMP-KDO synthetase), kds B, was previously cloned on a 9-kilobase Pst insert of Escherichia coli DNA into pBR 322 (Goldman, R. C., and Kohlbrenner, W. E. (1985) J. Bacteriol. 163, 256-261). Using a transposon mutagenesis approach we have now located kds B on this insert, which facilitated the isolation and sequencing of a 1.3-kilobase segment of DNA containing kds B and putative RNA polymerase and ribosome binding sites. The primary structure of CMP-KDO synthetase predicted by this nucleotide sequence was verified by amino acid composition and sequence analysis of purified CMP-KDO synthetase and cleavage fragments. Our results show that kds B consists of a 744-base open reading frame coding for a 248-amino acid peptide. The molecular weight of CMP-KDO synthetase calculated from the translated sequence is 27,486, taking into account the loss of the N-terminal methionine. These data define the transcriptional unit of kds B and its translation product in molecular terms, information prerequisite to our understanding of both the mechanism of CMP-KDO formation and the regulation of the KDO metabolic pathway in Gram-negative bacteria.

Chemical synthesis of a gene encoding the human complement fragment C5a and its expression in Escherichia coli.

A gene coding for the C5a fragment of the fifth component of human complement has been chemically synthesized, cloned, and expressed in Escherichia coli. The 253-base-pair gene fragment was built through a two-step enzymic assembly of 16 oligonucleotides, the average length of each being 32 residues. The oligonucleotides were synthesized by using the phosphoramidite method. The gene was cloned in a pBR322-derivative plasmid downstream from the lac up-promoter mutant, UV5-D. The expression of C5a was detected and measured by immunoassay and a radioligand binding assay. C5a from E. coli was comparable to C5a purified from human serum in inhibiting binding of human 125I-labeled C5a to its putative receptor on polymorphonuclear leukocytes. Studies of smooth muscle contraction in isolated guinea pig ileum showed that the recombinant C5a was biologically active and produced cross-tachyphylaxis with human serum-derived C5a. The results demonstrate the feasibility of expressing C5a anaphylatoxin in bacteria and provide a system for mutagenesis of the C5a protein.